We propose to develop a reliable and quantitative in vitro transformation system using mouse epidermal cells from skin tumor sensitive (SENCAR) and to use these cells plus those from tumor resistant mice to study the mechanism of action of chemical carcinogens and tumor promoters. Previous investigations by us on in vitro transformation of epidermal cells from Balb/c mice revealed that (1) a maximum of 20 percent of the exposed flasks containing epidermal cells gave rise to tumorigenic cell lines after a long latency period, (2) these transformed cells when injected into nude mice gave rise to anaplastic tumors and in some cases undifferentiated carcinomas, (3) we were unable to show a two-stage transformation system using phorbol ester tumor promoters. Although we began with a pure population of epidermal cells for the above in vitro studies, the cells appeared to dedifferentiate with time in culture and eventually die after 3 months. This we suspected was due to less than ideal culture conditions which did not allow the proper growth-differentiation balance that is found in vivo. After extensive investigations we found culture conditions that allowed the epidermal cells to maintain their epithelial morphology even after being subcultured several times. These cells have extensive desmosomes, produce keratin and stratify in vitro very similar to that of in vivo epidermis. These epidermal cells are grown on a 3T3 clone A31 feeder layer at 31 C in enriched Waymouth medium which is supplemented with 10 percent FCS with the addition of insulin (10 ug/ml), hydrocortisone (0.1 ug/ml) and epidermal growth factor (10ng/ml). Using transformation system using epidermal cells from skin tumor sensitive mice but also to develop a relevant one in which two-stage transformation is operational using phorbol ester tumor promoters and one in which the transformed cells give rise to keratinizing squamous cell carcinoma when injected into a nude mouse. At various times after the exposure of the epidermal cells to carcinogens or carcinogen-promoter the cells will be subjected to low serum levels or low calcium concentration in order to determine if this will allow an early selection of transformed cells.